The CD28-mediated costimulatory signal plays a pivotal role in the outcome of many immune responses including cytolytic responses in tumor and autoimmune diseases. Depending on the primary stimulation, CD28 can initiate multiple intracellular signaling pathways including signaling pathway that is insensitive towards immunosuppressive drug, Cyclosporin A (CsA). This CsA- insensitive pathway is believed to be involved in graft-vs-host disease (GVHD) during allogeneic bone marrow transplantation. Our current objectives are (1) to characterize the CsA-resistant costimulatory pathway; and (2) to determine the physiological significance of this signaling pathway in normal immune responses. Our recent work has demonstrated that the immunosuppressive drug rapamycin selectively affects the CsA-resistant pathway. Our initial studies have focused on the mechanism of activation of the IL-2 gene in a CsA-resistant manner. The effect of rapamycin on the IL-2 production was on the IL-2 mRNA stability. As the intracellular target of rapamycin is mTOR, we are investigating the mechanism of activation of mTOR by CD28 costimulation, and the role of mTOR in IL-2 mRNA stability. To examine the physiological role of this resistant pathway, we are studying the role of this pathway in the differentiation of T cells and allogeneic mixed lymphocyte reactions (MLR) as an in vitro model of GVHD. We propose to dissect the CsA- sensitive component of the allogeneic mixed lymphocyte reaction from the CsA-resistant one. We believe that further elucidation of this signaling pathway may assist in the identification of novel therapeutics to prevent GVHD.